Septic shock resulting from a systemic response to serious infection, e.g. gram-negative bacteremia, at local sites of infection or inflammation (e.g. the abdominal cavity) with the concomitant activation of the cytokine cascade, has been increasing in incidence over the last 50 years and is currently the commonest cause of death in intensive care units in the U.S. The reasons for this increase and high incidence of septic shock are believed to be the increased use of invasive devices such as intravascular catheters, increased use of cytotoxic and immunosuppressive drugs, increased longevity of patients liable to develop sepsis and an increase in infections caused by antibiotic-resistant organisms.
Disorders associated with sepsis are bacteremia (also known as septicemia) characterized by positive blood cultures; sepsis characterized by a systemic response to the infection in the form of tachypnea, tachycardia, hyperthermia or hypothermia; sepsis syndrome in which there is clinical evidence of sepsis and signs of altered organ perfusion in the form of an abnormally increased lactate level, oliguria or acutely altered mental status; early septic shock in which there is clinical evidence of sepsis syndrome as well as hypotension lasting for less than one hour and responsive to conventional therapy; and refractory septic shock in which there is clinical evidence of sepsis syndrome and hypotension lasting for more than one hour despite conventional therapy.
The continued high mortality and morbidity attributable to gram-negative sepsis has prompted an intensive search for therapeutic agents capable of counteracting the potentially lethal effects of circulating bacterial LPS. Numerous papers report a significant therapeutical effect of high doses of intravenously administered immunoglobulin. The treatment, however, requires IgG derived from the plasma of donors screened for naturally occurring high levels of antibodies to core LPS or from very large pools of donors (&gt;1000). Monoclonal antibodies against LPS which have also been suggested for the treatment of bacteremia (e.g. WO 88/03211) have shown little or no effect, probably because they do not inhibit the cytokine cascade induced by LPS. Furthermore, relatively few sepsis patients exhibit circulating endotoxemia and bacteremia so that antibodies neutralizing circulating LPS are not applied at the site where sepsis develops.
Mild oxidative stress is a normal feature in higher vertebrates as a result of a persistent stage of oxidative siege. Under normal conditions an efficient defense system consisting of an elaborate arsenal of antioxidants ensure that the organism is able to cope with oxygen free radicals by keeping a balance between oxygen free radicals and antioxidants. However, at sites of infection or injury, numerous aggressive oxidative species (oxygen free radicals) are secreted by phagocytes (activated neutrophil leukocytes, macrophages and monocytes) as a requisite to kill invading foreign pathogens causing infection. At these sites, the generation of oxygen free radicals is far beyond the antioxidant capacity of the surrounding cells, and these cell may be injured or die from necrosis or apoptosis (programmed cell death) mediated by oxygen free radicals.
At the site of infection or injury a cytokine cascade is initiated which, in turn, activates neutrophil leukocytes. The initiator of the cytokine cascade (in gram-negative bacteremia) is endotoxin (otherwise known as lipopolysaccharide, abbreviated to LPS) released at the infectious or inflammatory site where it induces the release of tumour necrosis factor .alpha. (TNF.alpha.), interleukin-1, interleukin-6,interleukin-8 and platelet-activating factor (PAF) from macrophages and other cells. After release of TNF.alpha., interleukin-1 and PAF, arachidonic acid is metabolized to form leukotrienes, thromboxane A.sub.2 and prostaglandins. Interleukin-1 and interleukin-6 activate T-cells to produce interferon-.gamma., interleukin-2, interleukin-4 and granulocyte-monocyte colony-stimulating factor. Neutrophils may be activated directly by most of these mediators. Neutrophil-induced damage may thus occur during degranulation by the release of oxygen free radicals and lysosomal enzymes, and during aggregation at infective or inflammatory sites.
Although the molecular mechanism responsible for LPS-mediated initiation of the cytokine cascade is not fully understood, recent reports of the signal transduction of the cytokines TNF.alpha., vitD.sub.3 and INF-.gamma. shed some light on the phenomenon.
The cytokines vitD.sub.3 and INF-.gamma. have been shown to stimulate production of ceramide in HL-60 cells by stimulating a membrane-bound neutral sphingomyelinase which hydrolyses membrane sphingomyelin to ceramide and phosphorylcholine (cf. T. Okazaki et al., J. Biol. Chem. 265, 1990, pp. 15823-15831). Ceramide has been found to be a second messenger which, in turn, activates a ceramide-activated protein kinase belonging to the family of X Ser/Thr Pro protein kinases (cf. S. Mathias et al., Proc. Natl. Acad. Sci. USA 88, 1991, pp. 10009-10013). Ceramide has additionally been shown to activate a ceramide-activated Ser/Thr protein phosphatase (cf. R. T. Dobrowski and Y. A. Hannun, J. Biol. Chem. 267, 1992, pp. 5048-5051). These initial reactions were shown to lead to further downstream signaling in a complex and as yet poorly understood manner, involving activation of the MAP kinase cascade, stimulation of transcription factors such as c-Myc and c-Fos, activation NF-KB and stimulation of PLA.sub.2 leading to the formation of arachidonic acid derivatives.
Lipoprotein-binding protein (LBP) in the circulation binds to LPS and mediates binding of LPS to the specific CD14 receptor. In a recent study of the signal transduction by LPS via the CD14 receptor on HL-60 cells, it was shown that LPS provokes its cellular responses, e.g. the initiation of the cytokine cascade by stimulation of the ceramide-activated protein kinase. Structural analysis has established that a portion of the reducing end of the lipid A moiety of LPS closely resembles a portion of ceramide (cf. C. K. Joseph et al., J. Biol. Chem. 269, 1994, pp. 17606-17610). It would therefore appear that LPS exerts its activity by entering into the ceramide pathway of cells.